Polymer, production and use thereof

ABSTRACT

The present invention provides a biodegradable high molecular polymer characterized in that the content of water-soluble low molecular compounds, as calculated on the assumption that each of said compounds is a monobasic acid, is less than 0.01 mole per 100 grams of said high molecular polymer.The thus-obtained high molecular polymer has good aging stability and can be used advantageously as an excipient for pharmaceutical preparations.

This application is a divisional of Ser. No. 858,040, filed 5/1/86, nowU.S. Pat. No. 4,728,721, filed 3/1/88.

This invention relates to a biodegradable (degradable in vivo) highmolecular polymer useful as an excipient in producing pharmaceuticalpreparations and a method of producing the same.

Biodegradable high molecular polymers may be used, for example, asexcipients for pharmaceutical preparations such as microcapsules. Asexamples of such biodegradable high molecular polymers, copolymers oflactic acid and glycolic acid are known to be obtainable bypolycondensation of lactic acid and glycolic acid in the presence of astrongly acidic ion exchange resin (cf. U.S. Pat. No. 4,273,920).

The present inventors also established that polymers or copolymers oflactic acid and/or glycolic acid may be obtained by polycondensation inthe presence of a solid inorganic acid catalyst or by poly-condensationwithout catalyst followed by removal of water and then polycondensation(cf. EPC Patent Publication (laid open) No. 0171907).

When produced by the methods so far used, biodegradable high molecularpolymers contain low molecular compounds such as an unreacted monomer ormonomers and polymers of low polymerization degree, so that when theyare used in producing microcapsules, incorporation rates intomicrocapsules of drugs to be microencapsulated are decreased or theso-called initial burst, namely extraordinarily initial drug releasefrom microcapsules after administration, tends to increase.

Furthermore, biodegradable high molecular polymers are chemicallyunstable. When allowed to stand at room temperature for several weeks toseveral months, they undergo degradation, which result in decrease inpolymerization degree.

In view of the above drawbacks, the present inventors treated saidbiodegradable high molecular polymers by a variety of methods and, as aresult, it was found that the content of water-soluble low molecularcompounds may be reduced by treating said polymers with water or amixture of water and an organic solvent readily soluble in water.Further investigation based on this finding has now led to completion ofthe present invention.

Thus, the invention provides a biodegradable high molecular polymercharacterized in that the content of water-soluble low molecularcompounds as calculated on the assumption that said compounds each are amonobasic acid is less than 0.01 mole per 100 grams of said highmolecular polymer, a method of producing the biodegradable highmolecular polymer which method comprises removing water-soluble lowmolecular compounds from a biodegradable high molecular polymercontaining not less than 0.01 mole of water-soluble low molecularcompounds per 100 grams thereof (as calculated on the assumption thatsaid compounds each is a monobasic acid) using water or a mixture ofwater and an organic solvent readily soluble in water, a microcapsulefor injectable sustained release containing ingredient and thebiodegradable high molecular polymer, and producing the same.

The biodegradable high molecular polymer to serve as the startingmaterial in performing the method of the invention may be produced byany method, for example by the method described in the above-cited U.S.Pat. No. 4,273,920 and EPC Patent Publication (laid open) No. 01719067.

Said starting material contains water-soluble low molecular compounds inan amount of not less than 0.01 mole per 100 grams thereof as calculatedon the assumption that each of said compounds is a monobasic acid.

The content of water-soluble low molecular compounds may be determinedby ordinary neutralization titration. Thus, for example, 300 mg of astarting high molecular compound is dissolved in 10 ml ofdichloromethane, the solution is stirred and shaken with 20 ml ofdistilled water for 10 minutes, the mixture is separated into an aqueousphase and an oily phase using a centrifuge, and the aqueous phase isassayed for free acids by neutralization titration using N/100 aqueousNaOH solution with phenolphthalein as an indicator. The number of molesof NaOH required for neutralization is converted to a free monobasicacid content.

The biodegradable high molecular polymer according to the presentinvention preferably has good biocompatibility and thus includes, amongothers, hydroxy acid polyesters (e.g. polylactic acid, polyglycolicacid), polycyanoacrylic acid esters, polyhydroxybutyric acid,poly-γ-caprolactone, polyorthoesters and polyorthocarbonates.

Said high molecular polymer may be a copolymer produced by using two ormore different monomers as the monomers for forming said high molecularpolymer. Said high molecular polymer may also be a block polymer or agraft polymer.

Among the high molecular polymers mentioned above, those degradable invivo at relatively high degradation rates are preferred.

Preferred examples of the high molecular polymer according to thepresent invention are polylactic acid and copolymers of lactic acid andglycolic acid. As the copolymers of lactic acid and glycolic acid,mention may be made of those comprising about 100-50 mole percent oflactic acid with the balance being glycolic acid.

Furthermore, those copolymers of lactic acid and glycolic acid whichhave a weight average molecular weight of about 2,000-50,000 arepreferred.

Further mention may be made of those copolymers of lactic acid andglycolic acid which are composed of about 90-50 mole percent of lacticacid and about 10-50 mole percent of glycolic acid and have a weightaverage molecular weight of about 5,000-35,000 and an inherent viscosityof about 0.05-0.5 dl/g as determined with a 0.5 weight percentchloroform solution thereof.

Examples of the organic solvent readily soluble in water which aresuited for use in carrying out the method of the present invention areacetone, methanol, ethanol, tetrahydrofuran, acetonitrile and ethylacetate. Among these, preferred from the safety viewpoint are acetoneand ethanol, and ethanol is more preferred.

When a mixture of water and such readily water-soluble organic solventis used, the water/organic solvent ratio (v/v) may be within the rangeof about 100/0 to 100/100, especially 100% water.

In carrying out the method of the invention, high molecular polymer asthe raw material is preferably dissolved in 3 to 20 time the amount(w/v) of an organic solvent [e.g. halogenated alkane (e.g.dichloromethane, chloroform, dichloroethane, trichloroethane), acetone,tetrahydrofuran, ethyl acetate, benzene] in advance to treatment by saidmethod since the use thereof in solution form is more efficient,although it may be used also in solid form (e.g. powder). Thus, whenbrought into contact with water or a mixture of water and an organicsolvent readily soluble in water, such high molecular polymerizationproduct dissolved in an organic solvent may acquire a markedly increasedcontact surface area upon application of stirring or some otherappropriate means.

The method according to the invention is conducted generally at atemperature of about 0°-90° C., preferably about 20°-70° C.

In accordance with the present invention, the biodegradable highmolecular polymerization product serving as raw material is mixed withwater or a mixture of water and an organic solvent readily soluble inwater with stirring to thereby remove water-soluble low molecularcompounds as a result of dissolution thereof in water or said mixture.Since the desired biodegradable high molecular polymer is insoluble inwater or said mixture said low molecular compounds may be separated fromthe desired high molecular polymer.

Although the ratio in quantity between water or a mixture of water andan organic solvent readily soluble in water and the high molecularpolymerization product serving as raw material is not critical for themethod according to the invention, it is desirable that water or themixture should be used in large excess. The treatment may also becarried out in a system equipped with an appropriate collecting meansand suited for continuous rinsing with water.

The above-mentioned stirring of water or the mixture may be effected bymeans of any of ordinary stirrers, shakers, blenders and the like. Meanshighly capable of causing sufficient mixing to remove unreacted materialor materials and water-soluble low molecular compounds from said highmolecular polymer to a satisfactory extent are desirable.

Since the desired high molecular polymer is not dissolved in water orsaid mixture but precipitates or separates, it may be recovered byseparating the precipitate, liquid droplets or solids by, for example,filtration or the like, and then drying the same.

By carrying out the method according to the invention, water-soluble lowmolecular compounds may be eliminated from the raw high molecularpolymerization product with good efficiency.

In purifying high molecular polymerization products in general, theprimary object is to remove catalysts, gaseous monomers, or highly toxicmonomers (e.g. vinyl chloride). In some instances, removal of lowmolecular compounds and/or unreacted materials is also intended, as inthe present invention. In such instances, the distillation method ismostly employed to thereby remove initial boiling fractions. However,removal of trace amounts of water-soluble substances is generallyunnecessary and, as a general rule, such method of removing trace amountof water-soluble substances as provided by the present invention isthought unnecessary and is not in practice.

The biodegradable high molecular polymer thus obtained has the followingfeatures:

(1) The high molecular polymer obtained by the method of the presentinvention as such or in pharmaceutical preparations produced by usingsaid high molecular polymer shows good stability in aging.

(2) When microcapsules are produced by using the high molecular polymerobtained by the method of the present invention w/o/w emulsionformation, followed by in-water drying, increased rates of drugincorporation in said microencapsulation are obtained.

(3) When microcapsules are produced by the method mentioned above in (2)using the high molecular polymer obtained by the method of the presentinvention, the initial burst (release by one day) of drugs frommicrocapsules is markedly reduced, so that the drugs are constantlyreleased over a prolonged period of time.

The biodegradable high molecular polymer obtained by the method of thepresent invention may be used, for instance, as an excipient formicrocapsules. Thus, for example, sustained release microcapsulescontaining a water-soluble ingredient, e.g. peptides, such as thosehaving luteinizing hormone releasing hormone-like activity and thyroidhormone releasing hormone-like activity, may be produced by preparing aw/o emulsion with a solution containing a water-soluble ingredientserving as the inner water phase, with a drug-retaining substance (mostpreferably gelatin, albumin, pectin, agar, or the like) added to theinner water phase as desired, and a solution containing thebiodegradable high molecular polymer obtained by the method of theinvention serving as the oil phase, dispersing said emulsion in a waterphase to give a w/o/w emulsion (preferably adjusting the viscosity ofthe w/o emulsion for preparing said w/o/w emulsion to about 150-10,000cp), and then the solvent in oil layer is desorbed. The thus-obtainedmicrocapsules may be administered as a sustained release injection. Thedose of such microcapsules may vary depending on the kind and content ofthe water-soluble active ingredient, the dosage form, the duration ofdrug release, the animal to be treated (e.g. warm-blooded mammal such asmouse, rat, horse, cattle or human) and the object of administration. Inany case, a dose is sufficient if it corresponds to the effective amountof said active ingredient. For instance, the dose may be suitablyselected within the range of about 0.02-200 mg/kg, preferably about0.2-40 mg/kg, of microcapsules per administration. In the use in asuspension form for the above-mentioned administration as an injection,the dose may be suitably selected within the range of about 0.1-5 ml,preferably about 0.5-3 ml, of the suspension.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 represents the changes with time in residual drug content in themicrocapsules obtained in Reference Example 3.

EXAMPLES

The following reference examples and working examples illustrate theinvention in further detail.

Reference Example 1

About 10 g of each of three lactic acid-glycolic acid copolymers (ratio75/25; average molecular weight 12,500) synthesized by different methods[i.e. (1) strongly anionic ion exchange resin catalyst method, (2) solidacid (acid clay) catalyst method and (3) catalystless method, each beinga polycondensation method described below]was dissolved in about 20 mlof dichloromethane and the solution was poured into 1,000 ml of hotwater at about 60° C. with stirring, whereby the dichloromethane wasevaporated and the high molecular polymer came up to the surface. Thelatter was collected and dried under reduced pressure for drying andsolvent removal to give the desired high molecular polymer. The polymerobtained was placed in a closed vessel and stored at room temperature.For stability evaluation, the thus stored sample was subjected to GPC(gel permeation chromatography) for average molecular weightdetermination. As shown by the results given in Table 1, markedimprovement in stability was noted with the high molecular polymersobtained in accordance with the invention and having low free monomeracid contents.

                                      TABLE 1                                     __________________________________________________________________________                        Average mol. wt.                                                                           Initial                                                              After storage                                                                          inherent                                     Method of  Lot.                                                                             Free acid (months) at room                                                                       viscosity                                    synthesis  No.                                                                              content(*)                                                                          Initial                                                                           temperature                                                                            dl/g                                         __________________________________________________________________________    (1)   Control                                                                            1-1                                                                              0.02  12,000                                                                            (12)                                                                               4,400                                                                             0.14                                               Invention                                                                          1-2                                                                              0.0033                                                                              11,900                                                                            (12)                                                                              11,600                                                                             0.14                                         (2)   Control                                                                            2-1                                                                              0.0132                                                                              12,500                                                                            (5)  4,100                                                                             0.15                                               Invention                                                                          2-2                                                                              0.0033                                                                              12,500                                                                            (5) 11,500                                                                             0.15                                         (3)   Control                                                                            3-1                                                                              0.0165                                                                              12,500                                                                            (5)  5,800                                                                             0.15                                               Invention                                                                          3-2                                                                              0.0055                                                                              12,500                                                                            (5) 12,000                                                                             0.15                                         __________________________________________________________________________

(*) Method of free acid determination: 300 mg of a sample is dissovledin 10 ml of dichloromethane, the solution is extracted with 20 ml ofdistilled water and 10 ml of the aqueous layer is titrated to neutralwith 0.01N NaOH (phenolphthalein indicator).

The values of free acid content given in Table 1 indicates the number ofmoles of free acids dissolved in water per 100 grams of the highmolecular polymer as calculated on the assumption that said free acidseach is a monobasic acid.

In preparing the lactic acid-glycolic acid copolymers used in the above,the following methods were used:

(1) Strongly anionic exchange resin catalyst method

To 160 g of 85% aqueous lactic acid solution and 38 g of glycolic acidwas added 6.8 g of Dowex 50W and the mixture was heated in a nitrogenatmosphere under reduced pressure for 6 hours in a manner such that theinside temperature and pressure were initially 105° C. and 350 mmHg,respectively, and finally 150° C. and 30 mmHg, respectively, whileremoving the water distilled. Then, 6.8 g of Dowex 50W was added and thereaction was further carried out at 175° C. and 3-5 mmHg for 40 hours.While hot, the reaction mixture was filtered to thereby remove the Dowex50W. The filtrate was cooled to give a lactic acid-glycolic acidcopolymer.

(2) Solid acid (acid clay) catalyst method

To 160 g of 85% aqueous lactic acid solution and 38 g of glycolic acidwas added 17.4 g of acid clay and the mixture was heated in a nitrogenatmosphere for 6 hours while increasing the temperature and degree ofpressure reduction stepwise in a manner such that the inside temperatureand pressure were initially 105° C. and 350 mmHg, respectively, andfinally 150° C. and 30 mmHg, respectively and while removing the waterdistilled. Thereafter, the inside pressure was reduced to 3 mmHg andheating was conducted for 36 hours while maintaining the insidetemperature at 175° C. The reaction mixture was cooled to roomtemperature, 400 ml of methylene chloride was added, the resultingmixture was stirred for dissolution of the polymerization product, theacid clay was then filtered off, and the filtrate was concentrated todryness to give a white lactic acid-glycolic acid copolymer.

(3) Catalystless method

To 160 g of 85% aqueous lactic acid solution was added 38 g of glycolicacid and the mixture was heated in a nitrogen atmosphere under reducedpressure for 6 hours in a manner such that the inside temperature andpressure were initially 105° C. and 350 mmHg, respectively, and finally150° C. and 30 mmHg, respectively, while removing the water distilled.Heating under reduced pressure was further conducted at 3-5 mmHg and175° C. for 36 hours. Upon cooling to room temperature, there wasobtained a colorless lactic acid-glycolic acid copolymer.

Reference Example 2

In 800 mg of distilled water were dissolved with warming 450 mg ofleuprolide [the acetate of a poly-peptide having the formula(Pyr)Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NH-CH₂ CH₃ and havingluteinizing hormone releasing hormone (LH-RH)-like activity, wherein theabbreviations according to the IUPAC-IUB Commission on Bio-chemicalNomenclature are used, the amino acids, unless otherwise specified,being in the L form] and 40 mg of gelatin (internal water phase).Separately, 3.5 g of each of the lactic acid-glycolic acid copolymers ofReference Example 1, Lot Nos. 2-1, 2-2, 3-1 and 3-3, was dissovled in 5ml of methylene chloride (oil phase). The oil phase was added to thewater phase with stirring using Polytron (Kinematica, Switzerland) togive a w/o emulsion. The viscosity at 15° C. of the w/o emulsionsderived from Lot No. 2-2 and No. 3-2 was 2,000. Separately, 200 ml of a0.5% aqueous solution of polyvinyl alcohol was prepared. To this wasadded the w/o emulsion with stirring using Autohomomixer (Tokushu Kita,Japan), whereby a (w/o)/w emulsion was produced.

This emulsion was stirred with a propeller in a nitrogen stream forabout 2 hours to thereby evaporate dichloromethane and to solidify theoil phase. The thus-formed microcapsules were collected by filtration,rinsed with water and dried. In 2 ml of dichloromethane and 7 ml ofdistilled water were dissolved 50 mg of the microcapsules obtained inthe powder form, and the leuprolide concentration in the distilled waterwas determined by reversed-phase EPLC and the content of leuprolideincorporated into the microcapsules was calculated. Said content isgiven in Table 2 in terms of percentage to the theoretical content.

                  TABLE 2                                                         ______________________________________                                                   Lot  Leuprolide content (%)                                        ______________________________________                                        Invention    2-2    95                                                        Invention    3-2    97                                                        Control      2-1    84                                                        Control      3-1    64                                                        ______________________________________                                    

As is evident from Table 2, the use of the high molecular polymersobtained by the method of the invention gave higher rates of leuprolideincorporation.

Reference Example 3

The microcapsules prepared in Reference Example 2 were weighed in 50-mgportions and each portion was dispersed in 10 ml of phosphate buffer (pH7.0). The release of leuprolide from the microcapsules into the bufferwas measured by stirring each dispersion at 25 rpm in aconstant-temperature vessel maintained at 37° C.

With the leuprolide content as found in Reference Example 2 taken as theinitial value, residual leuprolide percentages to the initial value weredetermined by subjecting the filtrates obtained after separation ofmicrocapsules by filtration after storage at 37° C. for 1, 7, 14, 21 and28 days to HPLC for determination of residual leuprolide. The percentagevalues thus obtained are shown in FIG. 1.

The data shown in FIG. 1 clearly indicate that the use of the highmolecular polymers according to the present invention reduced theinitial burst (release by one day) and allowed leuprolide release ofapproximately zero order over 1-1.5 months.

In FIG. 1, is for the high molecular polymer of Lot No. 2-1, for thehigh molecular polymer of Lot No. 2-2, o for the high molecular polymerof Lot. No. 3-1 and for the high molecular polymer of Lot No. 3-2.

Reference Example 4

A high molecular polymer was synthesized in the same manner as inReference Example 1, method (3). The free acid content was found to be0.021 mole per 100 g of the high molecular polymer obtained.

Reference Numeral 5

A high molecular polymer was produced by weighing 191 g of 85% aqueouslactic acid solution, 17.5 g of glycolic acid and 6.8 g of Dowex 50W andfollowing the procedure of Reference Example 1, method (1). Afterremoval of the water distilled, the reaction was performed at 3 mmHg and175° C. for 72 hours. In this case, the free acid content was 0.018 moleper 100 g of the high molecular polymer obtained.

Reference Example 6

By following the procedure of Reference Example 1, method (3), 150 g of85% aqueous lactic acid solution was treated and the water distilled wasremoved. Thereafter, the reaction was further conducted at 3 mmHg and175° C. for 12 hours to give a high molecular polymer. In this case, thefree acid content was 0.035 mole per 100 g of the high molecular polymerobtained.

Example 1

The polylactic acid-glycolic acid obtained in Reference Example 4 by thecatalystless method and having a lactic acid/glycolic acid ratio of75/25 and an average molecular weight of 13,000 was dissovled indichloromethane, and the solution was poured into hot water and about60° C. with stirring, whereupon a high molecular polymer came up to thesurface. This was collected and dried. The thus-obtained copolymer had alactic acid/glycolic acid ratio of 75/25, a molecular weight of 13,000and a free acid content of 0.005 mole per 100 g of the high molecularpolymer. Its inherent viscosity was 0.15 as determined in chloroform ata concentration of 0.5%.

Example 2

The polylactic acid-glycolic acid synthesized in Reference Example 5using Dowex 50W as catalyst and having a lactic acid/glycolic acid ratioof 90/10 and an average molecular weight of 20,000 was dissovled inacetone, and the solution was poured into warm water at about 40° C.,whereupon a high molecular polymer came up to the surface. The polymerwas collected and dried. The copolymer thus obtained had a free acidcontent of 0.008 mole per 100 g of the high molecular polymer and aninherent viscosity of 0.48 as determined in chloroform at aconcentration of 0.5%.

Example 3

The polylactic acid synthesized in Reference Example 6 without catalystand having an average molecular weight of 8,000 was finely pulverizedand then treated in warm water at 50° C. for 20 minutes with stirring,followed by filtration and drying. The thus-obtained high molecularcompound had a free acid content of 0.009 mole per 100 g thereof and aninherent viscosity of 0.10 as determined in chloroform.

Example 4

The same high molecular polymer as used in Example 1 was rinsed in a 1:1mixture of water and ethanol at 50° C. and then treated in the samemanner as in Example 1. The high molecular polymer obtained had a freeacid content of 0.0028 mole per 100 g of the high molecular polymer.

Example 5

In dichloromethane (oil phase) 3 g of lactic acid-glycolic acidcopolymer obtained in Reference Example 4 having lactic acid/glycolicacid ratio of 75/25 and an average molecular weight of 13,000 wasdissolved. 60 mg of thyroid hormone releasing hormone tartarate (TRH-T)was dissolved in 800 mg of water (inner water phase).

The oil phase was added to the inner water phase with stirring usingPolytron to give w/o emulsion. After cooling at 15° C., the w/o emulsionwas added to 200 ml of 0.5% aqueous solution of polyvinyl alcohol,separately prepared and cooled at 15° C., with stirring usingAutohomomixer to give (w/o)/w emulsion.

This emulsion was stirred with a propeller in a nitrogen stream forabout 2 hours to thereby cause evaporation of the dichhoromethane andsolidification of the oil phase. The thus formed microcapsules werecollected by filtration, rinsed with water and dried to form powder.

The "mole" used to show the weight amount of a compound is theabbreviation of "gram moles".

What is claimed is:
 1. A microcapsule for injectable sustained releasewhich contains an effective amount of ingredient and a biodegradablehigh molecular polymer useful as an excipient in producing apharmaceutical preparation comprising a copolymer or homopolymer ofabout 50-100 mole percent of lactic acid and about 50-0 mole percent ofglycolic acid having a weight average molecular weight of about 2,000 to50,000 and wherein the content of water-soluble low molecular compounds,as calculated on the assumption that each of said compounds is amonobasic acid, is less than 0.01 mole per 100 grams of said highmolecular polymer.
 2. The microcapsule according to claim 1, wherein theingredient is a water-soluble peptide.
 3. The microcapsule according toclaim 2, wherein the water-soluble peptide has luteinizing hormonereleasing hormone-like activity or thyroid hormone releasinghormone-like activity.
 4. The microcapsule according to claim 3, whereinthe peptide having the luteinizing hormone releasing hormone-likeactivity has the formula:

    (Pyr)Glu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHCH.sub.2 CH.sub.3.


5. The microcapsule according to claim 1, wherein the biodegradable highmolecular polymer has an inherent viscosity of about 0.05-0.5 dl/g asdetermined with a 0.5 weight percent chloroform solution thereof and aweight average molecular weight of about 5,000-35,000.